Journal Pre-proof

The expression profile of redox genes in human monocytes exposed in vitro to γ

radiation

Gina Manda, Cristian Postolache, Ionela Victoria Neagoe, Andreea Csolti, Elena
Milanesi, Maria Dobre
PII: S0969-806X(19)31262-9
DOI: https://doi.org/10.1016/j.radphyschem.2019.108634
Reference: RPC 108634

To appear in: Radiation Physics and Chemistry

Received Date: 28 September 2019

Revised Date: 30 November 2019
Accepted Date: 3 December 2019

Please cite this article as: Manda, G., Postolache, C., Neagoe, I.V., Csolti, A., Milanesi, E., Dobre, M.,
The expression profile of redox genes in human monocytes exposed in vitro to γ radiation, Radiation
Physics and Chemistry (2020), doi: https://doi.org/10.1016/j.radphyschem.2019.108634.

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 THE EXPRESSION PROFILE OF REDOX GENES
IN HUMAN MONOCYTES EXPOSED IN VITRO TO γ RADIATION

Gina Manda1*, Cristian Postolache2, Ionela Victoria Neagoe1, Andreea Csolti1, Elena Milanesi1,
Maria Dobre1

1
Victor Babes National Institute of Pathology, Splaiul Independentei Street, No. 99-101, 050096,
Bucharest, Romania
2
National Institute for Physics and Nuclear Engineering “Horia Hulubei”, Reactorului Street,
077125, Magurele, Romania

*Corresponding author:
Gina Manda, Victor Babes National Institute of Pathology, Splaiul Independentei Street, No. 99-
101, 050096, Bucharest, Romania, e-mail: gina.manda@gmail.com
Co-authors:
Cristian Postolache, National Institute for Physics and Nuclear Engineering “Horia Hulubei”,
Reactorului Street, 077125, Magurele, Romania, e-mail: cristip@nipne.ro
Ionela Victoria Neagoe, Victor Babes National Institute of Pathology, Splaiul Independentei
Street, No. 99-101, 050096, Bucharest, Romania, e-mail: neagoevictoria@gmail.com
Andreea Csolti, Victor Babes National Institute of Pathology, Splaiul Independentei Street, No.
99-101, 050096, Bucharest, Romania, e-mail: csoltiandreea@gmail.com
Elena Milanesi, Victor Babes National Institute of Pathology, Splaiul Independentei Street, No.
99-101, 050096, Bucharest, Romania, e-mail: elena.k.milanesi@gmail.com
Maria Dobre, Victor Babes National Institute of Pathology, Splaiul Independentei Street, No. 99-
101, 050096, Bucharest, Romania, e-mail: maria_dobre70@yahoo.com

HIGHLIGHTS
• 84 redox genes were investigated in human monocytes exposed in vitro to γ radiation;
• Important changes in the redox genes network were registered at 24-48 hrs post-
irradiation;
• Irradiated cells exhibit an enhanced potential to generate superoxide anion;
• NRF2 appears as a promising therapeutic target to protect irradiated normal monocytes.

1
ABSTRACT
Background It is known that γ radiation is rapidly triggering in cells and in the surrounding
medium a hydrogen peroxide burst through water radiolysis, which may have early and late
consequence on the viability and functions of irradiated cells. Investigations on monocytes and
macrophages are of utmost importance for assessing radiation risks, considering that they are
highly responsive to danger/damage signals, are master regulators of the immune response and
actively contribute to repair processes in tissues damaged by radiation.
Aim In the present study we investigated the expression changes of redox genes in SC
monocytes exposed in vitro to γ rays, aiming to identify delayed disturbances of the redox gene
network in exposed cells.
Methods SC monocytes were exposed to γ radiation in the dose range of 1-5 Gy, at various dose
rates (1.6 Gy/h, 4 Gy/h and 10 Gy/h). Cells were further cultivated and were analyzed at various
time points after irradiation (24 hrs and 48 hrs) regarding cell viability and the expression profile
of 84 redox genes critically involved in oxidative stress and antioxidant responses, addressing
ROS metabolism, oxidative stress responsive genes, antioxidant genes and pathway activity
signature genes.
Results Over-expression of particular genes encoding various members of the NADPH oxidase
family or myeloperoxidase evidenced an increased potential of irradiated cells to generate
reactive oxygen or nitrogen species, especially superoxide anion. Alternatively, up-regulation of
genes encoding antioxidant molecules indirectly proved that an increased oxidative activity
occurred in irradiated monocytes. Redox-mediated cellular deregulation was evidenced by the
over-expression of the BNIP3 gene which is involved in cell death by apoptosis or autophagy,
and was complemented by the up-regulation of the DUSP1 gene involved in cell cycle arrest and
functional silencing of monocytes. At lower doses and dose rates, an enhanced molecular
fingerprint of antioxidant responses mediated by the cytoprotective transcription factor NRF2
was found, albeit not being able to avoid the observed reduction of biabl cells in irradiated cell
cultures. Moreover, only a low transcriptional activity of NRF2 was registered at higher doses
which probably inflicted profound oxidative damages that compromised critical cellular defense
mechanisms.
Conclusion Pathway-focused analysis of redox gene expression allowed us to highlight delayed
oxidative alterations in monocytes exposed in vitro to γ rays, indicating that a chronic oxidative
activity is persisting long after irradiation. Moreover, we emphasized that the necessity to boost
the endogenous antioxidant system in normal cells exposed to higher doses of γ rays, in order to
protect them against the deleterious effects of radiation. A therapeutic option might be the
pharmacological activation of the transcription factor NRF2 which can rapidly react to even
small increases in intracellular oxidants and to regulate the expression of many antioxidant and
cytoprotective genes.

Key words: gamma rays, human monocytes, oxidative stress, redox genes, NRF2
Abbreviations: AA - arachidonic acid; ATCC - the American Type Culture Collection; ATP -
adenosine triphosphate; CD - cluster differentiation; COX2 - cyclooxygenase 2; DCA - dicentric
chromosome assay (DCA); FBS - Fetal Bovine Serum (FBS) = fetal calf serum (FCS); FC - fold
change; FDA - Food and Drug Administration (FDA); FR - fold regulation; GPx3 - glutathione
peroxidase 3; GSH - glutathione; GSSG - oxidized glutathione disulfide; HO-1 - heme

2
oxygenase 1; HT supplement - hypoxanthine-thymidine media supplement; IMDM - Iscove's
Modified Dulbecco's Medium; LDH - lactate dehydrogenase release test; MAPK - mitogen-
activated protein kinase; MKP1 - Mitogen-Activated Protein Kinase Phosphatase 1; MPO -
Myeloperoxidase; MTS - tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; NF-κB - nuclear factor
kappa-light-chain-enhancer of activated B cells; NO - nitric oxide; NRF2 - Nuclear factor
erythroid 2-related factor 2; OD - optical density; PBS - phosphate buffered saline; PTGS -
prostaglandin-endoperoxide synthase; qRT-PCR - Real-Time Quantitative Reverse Transcription
PCR; RNA - ribonucleic acid; RNS - reactive nitrogen species; ROS - reactive oxygen species;
SD - standard deviation; SEM - standard error of the mean; Trx - thioredoxin; TrxR - thioredoxin
reductase; UCP - mitochondrial uncoupling proteins;

3
1. INTRODUCTION
The biological effects of the exposure to ionizing radiation (accidental, occupational or
therapeutic) are not yet decisively defined and are still raising health concerns despite huge
progress in radiation physics and efficient shielding solutions. The first and foremost factor is the
total absorbed dose and the temporal pattern of radiation exposure. Other factors include
distribution of the radiation sources and structure and dimensions of the biological targets
(Howell, 2016). The health impact of lower doses is particularly challenging considering that
recent studies have evidenced that doses below 1 Sv may elicit persistent low-grade cellular
responses with important health detriments in the long run (Kamiya et al., 2015) related to cancer
(2015) and other pathologies, including vascular (Little, 2016) and neurological (Verreet et al.,
2016) disorders. Several lines of evidence have accumulated in recent years that the effects of
lower radiation doses may be less deleterious than expected from a linear extrapolation of the
radiation effects assessed at higher doses. We should be nevertheless aware that the assumption
of linear dose-effects, which is lately under scrutiny, may also underestimate low-dose risks in
particular cases (Brenner et al., 2003; Kamiya et al., 2015; Cardarelli and Ulsh, 2018). Therefore,
it is an urgent need to reconsider the biological risks posed by radiation exposure using the latest
concepts and tools of biomedical research in order to reshape radiation protection standards.
Reliable blood biomarkers should be identified and validated for setting up highly informative
epidemiological studies addressing a complex longitudinal evaluation of the health status of
professionally exposed workers (Lee, 2018). From the biomarkers perspective, the radiation-
triggered DNA damage and the associated repair processes (Mavragani et al., 2017) are generally
considered as main mechanisms underlining the biological impact of ionizing radiation. Different
biological approaches are applied for biodosimetry: the dicentric chromosome assay, the gamma-
H2AX foci assay or the cytokinesis block micronucleus assay (Rothkamm et al., 2013), but none
of these test is finally proved by regulatory authorities. Several large-scale studies have
investigated gene expression levels after irradiation. The DDB2, GADD45A, PCNA and PPM1D
genes related to the DNA repair and the regulatory JNK-p38 pathways were shown to play a
critical role in the human response to radiation exposure and share a lot of information with
radiation dose (Zhao et al., 2018). GADD45A provided the best discrimination of radiation dose
below and above 2 Gy, as assessed through extensive searches of the online databases from 1978
to 2017 (Lacombe et al., 2018). However, the variability of cellular response to different
qualities of radiation and temporal patterns still remains largely unknown, and additional
information is needed to understand the impact of these parameters on biomarkers.
Several lines of evidence have accumulated lately demonstrating that, in the case of low-dose
exposure, the damage inflicted by the radiation-triggered oxidative stress and its impact on
normal metabolism prevails over the biological consequences of DNA damage (Calabrese et al.,
2007; Vaiserman et al., 2018). More comprehensive understanding of the redox network in
irradiated cells is lately deriving from the finding that reactive oxygen species (ROS) at low
levels are highly needed for maintaining homeostasis through the complex network of redox
signaling events (Schieber and Chandel, 2014). Even small perturbations in the redox balance
(oxidants versus antioxidants) may functionally destabilize cells without inducing cell death, and
may have long-term health effects, if occurring on a chronic basis (Trachootham et al., 2008).
These observations need further documentation for in-depth characterization of the oxidative
activity and redox signaling in irradiated cells using a “network” approach and “multiplex”
investigation tools for drawing a comprehensive “connectome” map of redox processes elicited
by radiation exposure. However, standardization of experimental protocols and statistical
4
approaches is highly needed to increase the reproducibility of results and to overcome the large
discrepancy in the identified biomarkers.
In this context, the present study aims to investigate the expression profile of 84 genes critically
involved in oxidative stress and antioxidant responses of human SC monocytes exposed in vitro
to γ rays in various irradiation settings of dose and dose rate. Monocytes were addressed based
on the following considerations: a) monocytes and the corresponding tissue macrophages are
highly responsive to exogenous danger signals which are sensed through toll-like and NOD-like
receptors (Zhang and Mosser, 2008); b) monocytes are central regulators of the memory-bearing
adaptive immune response by shaping the cytokine profile and consequent functional
polarization of T lymphocytes towards a pro- or anti-inflammatory phenotype; c) tissue-resident
macrophages and newly recruited monocytes play an important role in tissue repair in the “injury
niche” (Ogle et al., 2016). The “network” approach applied in this study emphasized up-
regulation of several genes encoding for particular ROS sources, pointing towards an increased
oxidative activity mainly related to superoxide generation in irradiated cells. Additionally, the
over-expression of the redox-sensitive DUSP1 gene, which is known to induce cell cycle arrest,
was highlighted. Antioxidant mechanisms mediated by the cytoprotective NRF2 factor were
found over-expressed at ~1 Gy delivered at lower dose-rates, but could not limit the radiation-
induced decrease of viable SC cells, hence suggesting that pharmacologic activation of the NRF2
system might be a promising therapeutic strategy for counteracting the deleterious effects of γ
rays on blood monocytes.

2. MATERIALS AND METHODS

2.1. Cell cultures
Human SC monocytes (CRL-9855) from ATCC were cultivated in IMDM culture medium
(Gibco), supplemented with 10% fetal bovine serum (Biochrom), 1% antibiotic & antimycotic
solution (Sigma Aldrich, 10,000 units penicillin, 10 mg streptomycin and 25 µg amphotericin B
per mL) and 1% HT supplement (sodium hypoxanthine: 100 mM and thymidine 1.6 mM)
(Gibco), which will be further designated as complete culture medium. Cell cultures used in
experiments were mycoplasma free (certificate issued by Leibniz Institute DSMZ, Germany). SC
monocytes were maintained in culture by passages performed every 2 – 3 days to keep cellular
density in the range of (0.2 – 1) x 106 cells/mL. Cell cultures were monitored regarding cell
number and cell morphology by microscopic measurements and observations. Cells were used
for experiments between passages 4 and 12, when the multiplication rate was stable. 24 hrs
before irradiation, cell cultures were sub-cultured and brought to a density of 0.5x106 cells/mL.
Before experiments, cells were centrifuged and cellular density was adjusted to 5x106 cells/mL.
Cellular density was selected based on preliminary experiments showing that cells develop a
more stable and reproducible response to γ rays when they are irradiated at higher cellular
densities (data not shown). Transport of cells to the irradiation facility was performed at
temperatures in the range of 18-20oC.
2.2. Irradiation of SC cells
2.2.1. The γ irradiator
Irradiation of cell cultures with γ radiation was performed at the National R&D Institute for
Physics and Nuclear Engineering “Horia Hulubei”, Magurele, Romania, using a gamma
irradiator (Figure 1) with the following main characteristics was used: a) γ radiation fields were

5
produced by two 60Co sources with 4317 and 2760 TBq activity, placed inside of a hot cell for
radioprotection reasons; b) irradiation was performed horizontally and had a conical distribution
with an angle of approx. 30 degrees; c) the shielding, collimator and shutter were made of lead;
c) for maintaining a relatively uniform spatial density in samples during irradiation, an orbital
shaker, vertically arranged, with adjustable speed and timer, was used; d) cellular samples in
plastic cuvettes were placed in a support made of extruded polystyrene (23.2 g/L density),
positioned at distances over 10 cm from the orbital shaker plate to reduce the gamma retro-
reflection phenomena.

Figure 1. Schematic
representation of the γ irradiator

The dose rates used for exposure

of SC cells to γ radiation were adjusted according to: a) the activity of the 60Co source; b) the
distance between the samples and the 60Co source [D ~ f(1/x2)], and c) attenuation of gamma
radiation in lead layers [D = f(e-m x)]. D designates the dose and x the distance between samples
and the 60Co source.
The γ irradiator was analysed regarding: a) the ambient dose rate for different positions of the γ
irradiator, performed using an electronic debit-meter UNIDOS T10021 with Ionization Chamber
TM 30013; b) the axial and transverse dose rate distribution, using a dosimeter with alanine and
an ESR Spectrometer EMX micro-bay type (Kojima et al., 1993; Desrosiers et al., 2012) (Figure
2); c) the dose rate uniformity for different irradiation planes, using EBT3 Gafchromic films
(Sorriaux et al., 2013) (Figure 3).

6
 Figure 2. Axial and transverse dose rate distribution in the irradiation field, assessed, using a
dosimeter with alanine and an ESR Spectrometer EMX (micro-bay type)

Figure 3. The dose rate uniformity for different irradiation planes, assessed using EBT3
Gafchromic films an ESR Spectrometer EMX (micro-bay type)
2.2.2. Irradiation of SC monocytes
Irradiation of SC monocytes was performed using the γ irradiator described at 2.2.1. (Figure 1)
Cell suspensions (5x106 cells/mL) were placed in polystyrene cuvettes in the samples support,
and were introduced in the γ irradiator. Irradiation of cell suspension was performed under
shaking for maintaining a relatively uniform cell density in samples during irradiation. The
ambient temperature during irradiation was 18-19°C and therefore we may presume that cells had
low metabolic activity during irradiation as well as during samples transportation between the
cell culture facility and the irradiation facility. The dose uniformity in cellular samples was
assessed using EBT3 Gafchromic films placed in front and behind the samples support (see point
2.2.1.) and the obtained scanned images showed an uniform distribution of dose (Figure 3). The
deviation of the dose values from the irradiation axis was less than 7% in the area where the
biological samples were placed in the γ irradiator. The experimental results were confirmed by
GEANT4 simulations (Ioan et al., 2019). The dose rate distribution in the level of cellular
samples was analysed by assessing point doses using a dosimeter with alanine, and evidenced
parabolic distribution on the axial and transverse axis (Figure 2).

7
2.3. Post-irradiation investigations
After irradiation, cells were transported back to the cell culture facility at 18-20°C. They were re-
cultivated in T-25 culture flasks at a density of 0.5x106 cells/mL, using complete culture medium
(see point 2.2.1.). Cell cultures were monitored daily by microscopic investigations and cell
counting using a Burker-Turck counting chamber. The trypan blue exclusion test was used to
discriminate between live and dead cells, considering that dead cells will appear coloured in blue
when trypan enters into cells with compromised plasma membrane integrity.
At various time points post-irradiation part of the cell cultures were harvested for assessing
cellular viability and proliferation using the MTS reduction test (see point 2.3.1.) corroborated
with the lactate dehydrogenase (LDH) release test (see point 2.3.2.). These tests provide basic
information regarding the number of metabolically active cells and plasma membrane integrity,
respectively.
In parallel, cells were harvested, washed 2x in phosphate buffered saline (PBS), were processed
in TRIzol reagent (Thermo Fisher Scientific) and were stored at -80°C until use for RNA
extraction and gene expression investigations (see point 2.3.3.).
2.3.1. The MTS reduction test
The MTS reduction test was performed using the CellTiter 96® AQueous One Solution Cell
Proliferation Assay (Promega Corporation), according the manufacturer instructions. At the end
of the cultivation time, 20 µL of the kit reagent were added to each well. Cells were incubated
for 3 hrs at 37°C, in 5% CO2 atmosphere. The optical density (OD) of samples was measured at
490 nm against the 620 nm wavelength reference, using a Tecan Sunrise ELISA reader equiped
with the Tecan's Magellan universal reader control and data analysis software. Final OD in
cellular samples was calculated by subtracting the OD of the background acellular sample.
Results were presented as mean ± standard error of the mean (SEM) for OD triplicates per
sample.
2.3.2. The LDH release test
The LDH release test was performed using the CytoTox 96® Non-Radioactive Cytotoxicity
Assay (Promega Corporation), according the manufacturer instructions. Briefly, 50 µL of culture
supernatant were harvested and 50 µL of the kit reagent (LDH substrate) were added. Reaction
was allowed to develop for 30 min at room temperature in the dark, and was then stopped using
the stop solution provided in the kit. The optical density (OD) of samples was measured at 490
nm using a Tecan Sunrise ELISA reader equiped with the Tecan's Magellan universal reader
control and data analysis software. Final OD in cellular samples was calculated by subtracting
the OD of acellular background samples. Results were presented as mean ± SEM for OD
triplicates per sample.
2.3.3. Gene expression
Total RNA isolation from SC monocytes cells was performed using the TRIzol method. RNA
quality and quantity were assessed spectrophotometrically (NanoDrop 2000, Thermo Scientific)
based on the 260 nm /280 nm absorption ratio, with acceptable values ranging from 1.8 to 2.2.
Reverse transcription was performed with the RT2 First Strand Kit (Qiagen) using 600 ng RNA.
The expression of 84 genes critically involved in oxidative stress and antioxidant responses

8
(Table 1) was evaluated using the SYBR Green chemistry on the Stratagene Mx3005P
instrument (Thermo Fisher Scientific). The expression levels of each gene were normalized on
the geometric mean values of two housekeeping genes (RPLP0 and HPRT1) which were selected
based on the RefFinder algorithm (Xie et al., 2012) after the analysis of five candidate reference
genes (ACTB, B2M, GAPDH, HPRT1 and RPLP0), both in irradiated and non-irradiated cellular
samples (Figure 4).
To determine the Fold Change (FC) in gene expression (2^-∆∆CT), the normalized values (2^-
∆CT
exp) in irradiated cells was divided by the normalized expression in non-irradiated cells (2^-
∆CT
control). The Fold Regulation (FR) was reported as follows: when the FC value was above 1,
results have been reported as fold up-regulation; when the FC value was less than 1, the negative
inverse of FC have been reported as fold down-regulation.

Molecular pathway Gene Molecular pathway Gene

Antioxidants ROS metabolism

Glutathione GPX1, GPX2, GPX3, GPX,

Superoxide dismutases SOD1, SOD2, SOD3
peroxidases GPX5, GSTP1, GSTZ1
ALOX12, CCS, DUOX1,
PRDX1, PRDX2, PRDX3, Other superoxide
Peroxiredoxins DUOX2, MT3, NCF1, NCF2,
PRDX4, PRDX5, PRDX6 metabolism genes
NOS2, NOX4, NOX5, UCP2
CAT, CYBB, CYGB,
DUOX1, DUOX2, EPX, Other ROS metabolism AOX1, BNIP3, EPHX2,
Other peroxidases
LPO, MPO, PTGS1 , genes MPV17, SFTPD
PTGS2, TPO, TTN
ALB, APOE, GSR, MT3, APOE, ATOX1, CAT, CCL5,
VIMP, SOD1, SOD3, CYGB, DHCR24, DUOX1,
Other antioxidants
SRXN1, TXNRD1, DUOX2, DUSP1,
TXNRD2 EPX, FOXM1, FTH1,
GCLC, GCLM, GPX1,
GPX2, GPX3, GPX4,
GPX5, GSR, GSS,
Oxidative stress
HMOX1, HSPA1A, KRT1,
responsive genes
LPO, MBL2, MPO,
Oxygen
CYGB, MB MSRA, NQO1, NUDT1,
transporters
PDLIM1, PRDX2, PRDX5,
PRDX6 , PRNP, RNF7, VIMP,
SEPP1, SIRT2, SOD1, SOD2,
SQSTM1, SRXN1, TPO, TTN,
TXN, TXNRD1, TXNRD2

Table 1. The list of the investigated molecular pathways and redox genes contained in the RT²
Profiler™ PCR Array Human Oxidative Stress Plus (PAHS-065Y, Qiagen).

9
Figure 4. Comprehensive gene stability study performed using the RefFinder algorithm on five
candidate reference genes (ACTB, B2M, GAPDH, HPRT1 and RPLP0) both in irradiated and
non-irradiated cellular samples.
2.3.4. Statistics
Data from sample triplicates were presented as mean ± SEM. Data from independent
experiments were presented as mean ± SD (standard deviation). Comparison between groups
was performed using the Student’s T test.
3. RESULTS

3.1. The effects of γ radiation delivered at higher dose rates

Exposure of SC monocytes to γ rays (1.25, 2.5 and 5 Gy), delivered at a higher dose rate of 10
Gy/h, did not significantly alter MTS reduction and LDH release at 24 hrs post-irradiation
(Figure 5a). We should note the slightly but statistically significant increase of MTS reduction by
cells exposed to γ rays (140 ± 3)% p<0.05). In parallel, LDH release data did not show any
significant changes in irradiated cells against non-irradiated ones, indicating that the plasma
membrane integrity was not affected. It is possible that exposure of cells to γ rays might activate
at 24 hrs after irradiation particular metabolic enzymes that are underlining the reduction of MTS
such as dehydrogenases, in an attempt of the irradiated cells to increase their metabolism for
counteracting the deleterious effects of γ irradiation.
Obvious harmful effects of γ irradiation on the viability and proliferation of SC cells were
registered later, at 48 hrs post-exposure. A dose-independent decrease of the MTS reduction was
evidenced (Figure 5a), and was accompanied by a linear and dose-dependent increase of LDH
release (y = 0.0952x + 0.2568, R² = 0.98) (Figure 5b). The obtained data on MTS reduction were
negatively correlated with the paired data on LDH release (r = - 0.75, p = 0.0042). These results
10
indicate a decrease in the number of metabolically active cells that was partially due to cell death
by necrosis/necroptosis (cell death forms that are characterized by compromised plasma
membrane integrity, hence allowing intracellular proteins like LDH to be released outside the
cell). Our results do not rule out that apoptotic cell death occurs, even earlier than necrosis.

MTS reduction by SC monocytes

exposed to γ radiation
0.35
0.30
0.25
* * *
OD

0.20
24 hrs
0.15 * * *
48 hrs
0.10
0.05
0.00
0 1.25 2.5 5
Dose (Gy)

a)

LDH release by SC monocytes

exposed to γ raydiation
0.7 ***
0.6 ***

0.5 *
OD

0.4
24 hrs
0.3
48 hrs
0.2
0.1
0
0 1.25 2.5 5
Dose (Gy)

b)
Figure 5. MTS reduction and LDH release by SC monocytes exposed to γ radiation at a dose
rate of 10 Gy/h. Cells were further cultivated and analyzed at 24 hrs and 48 hrs post-exposure.
(a) MTS reduction by irradiated and non-irradiated SC monocytes; (b) LDH release by irradiated
and non-irradiated SC monocytes. Results are presented as mean ± SEM for triplicate samples.
Comparison between irradiated and non-irradiated samples was performed: *p<0.05,
***p<0.001.

11
 Related pathways
Pathway Gene Role of the encoded protein
Gene Ontology (GO)
The encoded protein is a phosphatase with dual specificity for
tyrosine and threonine. It can dephosphorylate the ERK2
Related pathways:
mitogen-activated protein kinase (MAPK), which results in
• RET signaling
the modulation of several cellular processes. This protein
• Immune response Fcepsilon RI
Dual specificity appears to play an important role in the human cellular
DUSP1 pathway
phosphatase 1 response to environmental stress as well as in the negative
GO annotations: phosphatase activity and
regulation of cellular proliferation. The encoded protein can
protein tyrosine/serine/threonine
make some solid tumors more resistant to both chemotherapy
Proliferation phosphatase activity
and radiotherapy, and therefore is a promising therapeutic
and cell death target in cancer.
Related pathways:
Encodes a mitochondrial protein that contains a BH3 domain • Legionellosis
BCL2 and acts as a pro-apoptotic factor. The encoded protein • HIF-1-alpha transcription factor
BNIP3 interacting interacts with anti-apoptotic proteins, including the E1B network
protein 3 protein and Bcl2. This gene is silenced in tumors by DNA GO annotations: protein
methylation. homodimerization activity and protein
heterodimerization activity
Prostaglandin-endoperoxide synthase (PTGS), also known as
Related pathways:
cyclooxygenase, is the key enzyme in prostaglandin
• IL-1 Family Signaling Pathways
biosynthesis, and acts both as a dioxygenase and as a
• Etoposide Pathway,
Prostaglandin- peroxidase. There are two isozymes of PTGS: a constitutive
Pharmacokinetics/Pharmacodyna
Inflammation PTGS2 endoperoxide PTGS1 form and an inducible PTGS2 form, which differ in
mics
synthase 2 their expression regulation and tissue distribution. This gene
GO annotations: protein
encodes the inducible isozyme and it is regulated by specific
homodimerization activity and lipid
stimulatory events, suggesting involvement in prostanoid
binding
biosynthesis and hence in inflammation and mitogenesis.
The encoded protein is a 47 kDa cytosolic subunit of Related pathways:
neutrophil NADPH oxidase (p47-phox). This oxidase is a • Signaling by Rho GTPases
Neutrophil
NCF1 multi-component enzyme that is activated to produce • RET signaling
cytosolic factor 1
superoxide anion. Mutations in this gene have been GO annotations: SH3 domain binding and
associated with chronic granulomatous disease. phosphatidylinositol binding
ROS sources
This gene encodes neutrophil cytosolic factor 2, the 67-kDa
Related pathways:
cytosolic subunit (p67-phox) of the multi-protein NADPH
• Signaling by Rho GTPases
Neutrophil oxidase 2 complex that is found in neutrophils. This oxidase
NCF2 • RET signaling
cytosolic factor 2 produces a burst of superoxide which is delivered to the
GO annotations: protein C-terminus
lumen of the phagosome. Mutations in this gene as well as in
binding and Rac GTPase binding
other NADPH oxidase subunits, can result in chronic

12
 granulomatous disease, a disease that causes recurrent
infections. Alternative splicing results in multiple transcript
variants encoding different isoforms.
The encoded protein is a glycoprotein and a member of the
NADPH oxidase family. It contains an iodide transporter,
thyroperoxidase and a peroxide generating system that
includes proteins encoded by this gene and the similar Related pathways:
DUOX2 gene. It is a dual oxidase because it has both a • Viral mRNA Translation
DUOX1 Dual oxidase 1 peroxidase homology domain and a gp91phox domain. The • Amine-derived hormones
protein generates hydrogen peroxide and thereby plays a role GO annotations: calcium ion binding and
in the activity of thyroid peroxidase, lactoperoxidase and in heme binding
lactoperoxidase-mediated antimicrobial defense at mucosal
surfaces. Two alternatively spliced transcript variants
encoding the same protein have been described for this gene.
This gene is predominantly expressed in the testis and
lymphocyte-rich areas of the spleen and lymph nodes. It Related pathways:
encodes a calcium-dependent NADPH oxidase that generates • Apoptotic Pathways in Synovial
NADPH oxidase superoxide and functions as a calcium-dependent proton Fibroblasts
NOX5
5 channel that may regulate redox-dependent processes in • PAK Pathway
lymphocytes and spermatozoa. Alternatively spliced GO annotations: calcium ion binding and
transcript variants encoding different isoforms have been heme binding
described for this gene.
Myeloperoxidase (MPO) is a heme protein synthesized
during myeloid differentiation that constitutes the major Related pathways:
component of neutrophil azurophilic granules. Produced as a • Etoposide Pathway
single chain precursor, myeloperoxidase is subsequently Pharmacokinetics/Pharmacodyna
MPO Myeloperoxidase cleaved into a light and heavy chain. The mature mics
myeloperoxidase is a tetramer composed of 2 light chains and • Phenytoin Pathway
2 heavy chains. This enzyme produces hypohalous acids and GO annotations: chromatin binding and
the greatly contributes to the microbicidal activity of heme binding
neutrophils.
Related pathways:
Nitric oxide is a reactive free radical which acts as a biologic
• IL-1 Family Signaling Pathways
mediator in several processes, including neurotransmission
• Immune response IL-23
Nitric oxide and anti-microbial as well as anti-tumoral activities. This
RNS sources NOS2 signaling pathway
synthase 2 gene encodes a nitric oxide synthase which is expressed in
GO annotations: protein
liver and is inducible by a combination of lipopolysaccharide
and certain cytokines also in monocytes and macrophages. homodimerization activity and
oxidoreductase activity
Heme oxygenase Heme oxygenase, an essential enzyme involved in heme Related pathways:
NRF2 targets HMOX1
1 catabolism, cleaves heme to form biliverdin, which is • IL-1 Family Signaling Pathways

13
 subsequently converted to bilirubin by biliverdin reductase, • IL-10 Pathway
and carbon monoxide, a putative neurotransmitter. Heme GO annotations: protein
oxygenase activity is induced by its substrate heme and by homodimerization activity and
various non-heme substances. Heme oxygenase occurs as two oxidoreductase activity.
isozymes, an inducible heme oxygenase-1 and a constitutive
heme oxygenase-2. HMOX1 and HMOX2 belong to the heme
oxygenase family.
This gene is a member of the NAD(P)H dehydrogenase
(quinone) family and encodes a cytoplasmic 2-electron
reductase. This FAD-binding protein forms homodimers and
reduces quinones to hydroquinones. This protein's enzymatic
activity prevents the one electron reduction of quinones that Related pathways:
NAD(P)H results in the production of radical species. Mutations in this • Acetaminophen Pathway,
NQO1 quinone gene have been associated with tardive dyskinesia, an Pharmacokinetics
dehydrogenase 1 increased risk of hematotoxicity after exposure to benzene • Ubiquinol biosynthesis
and susceptibility to various forms of cancer. Altered GO annotations: oxidoreductase activity
expression of this protein has been seen in many tumors and
is also associated with Alzheimer's disease. Alternate
transcriptional splice variants, encoding different isoforms,
have been characterized.
This gene encodes a member of the class-I pyridine
nucleotide-disulfide oxidoreductase family. The enzyme is a
homodimeric flavoprotein and has an important role in Related pathways:
Glutathione- cellular antioxidant defense by reducing oxidized glutathione • Amino Acid metabolism
GSR disulfide disulfide (GSSG) to the sulfhydryl form (GSH), the later one • Viral mRNA Translation
reductase being an important cellular antioxidant. Rare mutations in GO annotations: oxidoreductase activity
this gene result in hereditary glutathione reductase and flavin adenine dinucleotide binding
deficiency. Multiple alternatively spliced transcript variants
encoding different isoforms have been found.
This gene encodes a multifunctional protein that binds
ubiquitin and regulates activation of the nuclear factor kappa- Related pathways:
B (NF-kB) signaling pathway. The protein functions as a • TNF signaling (REACTOME)
scaffolding/adaptor protein in concert with TNF receptor- • Innate Immune System
SQSTM1 Sequestosome 1
associated factor 6 to mediate the activation of NF-kB in GO annotations: protein
response to upstream signals. Alternatively spliced transcript homodimerization activity and protein
variants encoding either the same or different isoforms have serine/threonine kinase activity
been identified for this gene.
This gene encodes a member of the sirtuin family of proteins, Related pathways:
SIRT2 Sirtuin 2 homologs to the yeast Sir2 protein. Members of the sirtuin • p53 signaling
family are characterized by a sirtuin core domain and • Chromatin Regulation

14
 grouped into four classes. The functions of human sirtuins /Acetylation
have not yet been fully determined; however, yeast sirtuin GO annotations: chromatin binding and
proteins are known to regulate epigenetic gene silencing and histone deacetylase binding
suppress recombination of DNA. Studies suggest that the
human sirtuins may function as intracellular regulatory
proteins with mono-ADP-ribosyltransferase activity. The
protein encoded by this gene is included in class I of the
sirtuin family. Several transcript variants result from
alternative splicing of this gene.
Related pathways:
Sulfiredoxin 1 contributes to oxidative stress resistance by
• Photodynamic therapy-induced
reducing cysteine-sulfinic acid formed under exposure to
NFE2L2 (NRF2) survival
oxidants of particular peroxiredoxins 1-4, but does not act on
SRXN1 Sulfiredoxin 1 signaling
peroxiredoxin 5 or peroxiredoxin 6. It catalyzes the reduction
GO annotations: oxidoreductase activity,
in a multi-step process by acting both as a specific
acting on a sulfur group of
phosphotransferase and a thioltransferase.
donors and sulfiredoxin activity
The encoded gene is a member of the peroxiredoxin family of
antioxidant enzymes, which reduce hydrogen peroxide and
Related pathways:
alkyl hydroperoxides. It may play a protective antioxidant
• Prostaglandin 2 biosynthesis and
role in cells, and may contribute to the antiviral activity of
PRDX1 Peroxiredoxin 1 metabolism
CD8+ T lymphocytes. This protein may have a pro-
• Peroxisome
proliferative effect and play a role in cancer development and
progression. Four transcript variants encoding the same GO annotations: identical protein binding
protein have been identified for this gene.
The protein encoded by this gene acts as a homodimer which Related pathways:
is involved in many redox reactions. The encoded protein is • TNF Signaling (REACTOME)
found in the cytoplasm and is active in the reversible S- • Innate Immune System
TXN Thioredoxin
nitrosylation of cysteines in certain proteins, which is part of GO annotations: oxidoreductase activity,
the response to intracellular nitric oxide (NO). Two transcript acting on a sulfur group of donors,
variants encoding different isoforms have been found. disulfide as acceptor
The encoded protein belongs to the pyridine nucleotide-
TXN metabolism disulfide oxidoreductase family, and is a member of the
thioredoxin (Trx) system. Three thioredoxin reductase (TrxR)
Related pathways:
isozymes are found in mammals. TrxRs are selenocysteine-
• Pathways in cancer
Thioredoxin containing flavoenzymes which reduce thioredoxins, as well
TXNRD2 • PAK Pathway
reductase 2 as other substrates, and play a key role in redox
homoeostasis. The gene encodes a mitochondrial form that is GO annotations: oxidoreductase activity
important for scavenging ROS in mitochondria. It functions and NADP binding
as a homodimer containing FAD and has a selenocysteine in
the active site. Alternatively spliced transcript variants

15
 encoding different isoforms, including a few ones that are
localized in the cytosol and some lacking the C-terminal Sec
residue, were found for this gene.
This gene encodes a member of the iron/manganese
Related pathways:
superoxide dismutase family, a mitochondrial protein that
• Respiratory electron transport,
forms a homotetramer and binds one manganese ion per
ATP synthesis by chemiosmotic
subunit. This protein binds to the superoxide resulting as
coupling, and heat production by
Superoxide byproduct of oxidative phosphorylation, and converts it to
SOD2 uncoupling proteins.
dismutase 2 hydrogen peroxide and molecular oxygen. Mutations in this
• Detoxification of Reactive
gene have been associated with idiopathic cardiomyopathy,
Oxygen Species
premature aging, sporadic motor neuron diseases as well as
GO annotations: identical protein binding
cancer. Alternative splicing of this gene results in multiple
and oxygen binding
transcript variants.
Mitochondrial uncoupling proteins (UCP) are members of the
larger family of mitochondrial anion carrier proteins. UCPs
separate oxidative phosphorylation from ATP synthesis with
energy dissipated as heat, also referred to as the
Related pathways :
mitochondrial proton leak. UCPs facilitate the transfer of
• Respiratory electron transport,
anions from the inner to the outer mitochondrial membrane
Uncoupling ATP synthesis by chemiosmotic
UCP2 and the return transfer of protons from the outer to the inner
protein 2 coupling, and heat production by
mitochondrial membrane. They also reduce the mitochondrial
Other uncoupling proteins
membrane potential in mammalian cells. Tissue specificity
cytoprotectors occurs for the different UCPs. The exact mechanisms through • Glucose /Energy Metabolism
which UCPs transfer H+/OH- are not known. This gene is
expressed in many tissues, with the highest expression in
skeletal muscle.
The encoded protein belongs to the glutathione peroxidase
family. These enzymes catalyze the reduction of organic
hydroperoxides and hydrogen peroxide by GSH, and thereby
protect cells against oxidative damage. Several isozymes of
Related pathways:
this gene family exist in vertebrates, which vary in cellular
• Detoxification of Reactive
location and substrate specificity. This particular isozyme is
Glutathione Oxygen Species
GPX3 secreted and is abundantly found in plasma. Down-regulation
peroxidase 3 • Selenium Micronutrient Network
of the expression of this gene by promoter hypermethylation
GO annotations: transcription factor
has been observed in a wide spectrum of human
binding and selenium binding
malignancies, including thyroid cancer, hepatocellular
carcinoma and chronic myeloid leukemia. This isozyme is a
selenoprotein. Alternatively spliced transcript variants have
been found for this gene.
HSPA1A Heat shock This intronless gene encodes a 70kDa heat shock protein Related pathways:

16
 protein family A which is a member of the heat shock protein 70 family. In • CDK-mediated phosphorylation
(Hsp70) member conjunction with other heat shock proteins, this protein and removal of Cdc6
1A stabilizes existing proteins against aggregation and mediates • Apoptosis Modulation and
the folding of newly translated proteins in the cytosol and in Signaling
organelles. It is also involved in the ubiquitin-proteasome GO annotations: ubiquitin protein ligase
pathway through interaction with the AU-rich element RNA- binding
binding protein 1. The gene is located in the major
histocompatibility complex class III region, in a cluster with
two closely related genes which encode similar proteins.
Table 2. The role of the genes with modified expression registered in γ irradiated SC monocytes (Adapted after GeneCards,
https://www.genecards.org/)

17
The expression of 84 redox genes (Table 1) was investigated by qRT-PCR in irradiated cellular
samples versus non-irradiated ones. The redox genes expression profile showed particular
changes induced by γ irradiation at 24 hrs and 48 hrs post-exposure of SC monocytes. The role
of these genes is presented in Table 2.
At 24 hrs post-irradiation of SC monocytes (-1.5≤ FR >1.5) significant expression changes
(Figures 6 and 7) for several of the investigated redox genes were registered at all investigated
doses (1.25, 2.50 and 5 Gy), as presented in Figure 6.
DUSP1 and BNIP3 genes were found over-expressed in irradiated SC cells as compared to non-
irradiated controls at all investigated doses (FR=2.42±0.65 and 1.93±0.27, respectively). DUSP1
encodes the dual specificity protein phosphatase 1 MKP-1 which is limiting cell proliferation as
well as the priming, migration and recruitment of monocytes (Kim et al., 2012). Consequently,
MKP-1 allows cells to repair oxidative damages, including those induced by exposure of cells to
ionizing radiation, and to avoid aberrant activation of monocytes. Moreover, over-expression
BNIP3 confers to irradiated monocytes increased susceptibility to apoptosis or autophagy. It is
contributing therefore to the elimination of oxidatively-damaged cells, but may also sustain the
survival of stressed cells by preventing ATP depletion or by eliminating damaged mitochondria
(Tracy and Macleod, 2007). The potential functional consequences of the increased expression of
DUSP1 and BNIP3 genes in irradiated SC cells were detected in fact later in our experiments, at
48 hrs after exposure of SC cells to γ rays. At this time point, the number of metabolically active
cells (evaluated by the MTS reduction test) was found to be significantly decreased in
comparison with non-irradiated controls (Figure 5a), but this may be due also to other
mechanisms than those mediated by DUSP1 and BNIP3 .
Analysis of the expression pattern of genes encoding ROS sources at 24 hrs after irradiation of
SC monocytes showed that there is no consistent up-regulation of the genes encoding ROS
sources such as NADPH-oxidases (CYBB, NCF1, NCF2) or myeloperoxidase (MPO gene) over
all investigated doses (Figure 7). Nevertheless, it is notable that an over-expression of genes
involved in superoxide production was evidenced at the irradiation dose of 5 Gy (CYBB:
FR=1.99, NCF1: FR=1.93, NCF2: FR=1.5). This may contribute to an increase of the basal
oxidative activity in monocytes exposed to higher doses of γ radiation, hence probably
accounting for the observed cytotoxic consequences of irradiation, as shown by the MTS
reduction (Figure 5a) and LDH release data (Figure 5b).
SC monocytes responded at 24 hrs post-irradiation with an enhanced molecular signature of the
cytoprotective transcription factor NRF2, as observed from the over-expression at all
investigated doses of two of its main targets, namely the GCLC (FR=-2.05±0.12) and the
HMOX1 gene (FR=-1.88±0.30) (Figure 7). GCLC encodes the glutamate-cysteine ligase catalytic
subunit that is involved in the first steps of glutathione biosynthesis, the most abundant cellular
antioxidant. HMOX1 encodes heme oxygenase 1 (HO-1) that catalyzes the degradation of heme
and mediates a large panel of cytoprotective effects (Loboda et al., 2016). Other NRF2 targets
were found over-expressed only at higher doses (2.5 Gy and 5 Gy) when irradiated cells were
probably exposed to higher oxidants levels, more or less directly elicited in SC monocytes by the
γ radiation. It is the case of the glutathione S reductase gene (GSR, FR=1.90±0.27) and the
glutathione peroxidase 2 gene (GPX2, FR=1.99±0.37) (Figure 7) that are both involved in
glutathione metabolism by controlling its redox status and cycling between the oxidized and
reduced form (Wu et al., 2004). The transcriptional signature of NRF2 in irradiated cells was a
clear indicative of enhanced oxidative activity in irradiated SC cells, considering that NRF2

18
activation and consequent antioxidant responses are elicited by oxidation of critical cysteines in
the NRF-KEAP1 system. The observed molecular changes appeared to be most prominent at
higher doses (2.5 Gy and 5 Gy) when an over-expression of superoxide sources was evidenced at
gene expression level (Figure 7.
Irradiation also induced an increase in arachidonate (AA) metabolism in SC monocytes at 24 hrs
post-exposure to γ rays, as shown by the over-expression of particular redox genes involved in
lipid metabolism: the PTGS2 gene (FR=1.24±0.56) that was found over-expressed at all
investigated doses (Figures 6 and 7), and the ALOX12 gene (FR=2.23±0.36) which was found
up-regulated only at the higher investigated doses (2.5 Gy and 5 Gy) (Figure 6). Up-regulation of
these redox genes is probably triggered by the radiation-induced oxidative burst, mainly through
the contribution of the superoxide generated by NADPH oxidases (Barbieri et al., 2003; Laube et
al., 2016; Gerrick et al., 2018). PTGS2 encodes for the inducible form of cyclooxygenase
(COX2) that triggers prostanoid biosynthesis with functional consequences on apoptosis
resistance and functional polarization of monocytes towards a pro-inflammatory M1 phenotype
(Martinez et al., 2006). Meanwhile, ALOX12 encodes the 12-lipooxygenase isoform that
mediates eicosanoid biosynthesis and is involved in the polarization of monocytes towards an
anti-inflammatory M2 phenotype (Martinez et al., 2006).
At 48 hrs after irradiation the gene expression profile of SC monocytes showed a distinctive
pattern as compared to the profile registered at 24 hrs post-exposure (Figure 6). The only genes
with consistently modified expression at 48 hrs post-irradiation, registered at all investigated
doses, were: a) the NOX5 gene (FR=3.47±1.87) encoding NOX5, a particular NOX isoform
shown to be constitutively expressed in monocytes and to contribute to enhanced oxidative stress
in various pathologic conditions (Manea et al., 2015); b) the GPX3 gene encoding glutathione
peroxidase 3 (FR=-4.43±1.41) which catalyzes the reduction of organic hydroperoxides and
hydrogen peroxide by glutathione, thereby protecting cells against oxidative damage. At higher
doses (2.5 Gy and 5 Gy) some other ROS and reactive nitrogen species (RNS) sources were
found to be over-expressed, as follows. DUOX1, which encodes a dual phosphatase enzyme that
produces hydrogen peroxide, was found over-expressed (FR=1.61±0.12) at 2.5 and 5 Gy, while
the DUOX2 isoform was over-expressed only at 5 Gy (FR=2.24). The NOS2 gene encoding the
inducible form of nitric oxide synthase (iNOS or NOS2) was also up-regulated (FR=2.33±0.14)
at 2.5 Gy and 5 Gy, indicating a potentially enhanced nitrostative stress in SC monocytes at 48
hrs post-exposure to γ radiation. Moreover, enhanced gene expression of both superoxide and
NO cellular sources may lead to increased formation of the highly reactive and toxic
peroxynitrite.

19
 24 hrs post-irradiation 48 hrs post-irradiation
Molecular Pathway Gene
1.25 Gy 2.50 Gy 5.00 Gy 1.25 Gy 2.50 Gy 5.00 Gy
DUSP1
Proliferation & cell death
BNIP3
Lipid metabolism & ALOX12
inflammation PTGS2
NOX5
DUOX1
DUOX2
ROS sources
NCF1
NCF2
MPO
RNS sources NOS2*
HMOX1

NRF2 targets GCLC

GSR
DHCR24
NQO1
GPX3
SOD1
Other antioxidants
SOD2
SOD3*
Heat shock proteins HSPA1A
Figure 6. Heatmap chart illustrating the expression level of redox genes in SC monocytes
exposed to various doses of γ radiation delivered at 10 Gy/h, assessed by qRT-PCR at 24 hrs and
48 hrs post-irradiation
Colour code:
FR≥6
4<FR<6
2≤FR≤4
1.5<FR<2
-2<FR<-1.5
-4<FR≤-2
FR≤-4
* genes with very low expression level in non-irradiated SC cells.

20
 3.5
3.0
2.5
2.0
Fold regulation (FR)

1.5
1.0
0.5
0.0
BNIP3 DUSP1 PTGS2 NOS2 GCLC HMOX1
-0.5
-1.0
-1.5
-2.0

Figure 7. Mean expression level of particular redox genes exhibiting in SC monocytes exposed γ
rays (delivered at a dose rate of 10 Gy/h) significant up- or down-regulation (-1.5≤ FR >1.5) at
all investigated doses (1.25, 2.5 and 5 Gy). Gene expression was analyzed at 24 hrs post-
irradiation by qRT-PCR using a pathway-focused array. Results are expressed as fold regulation
(FR) and are presented as mean ± SD for triplicate samples.
No significantly enhanced transcriptional signature of the transcription factor NRF2 was detected
in irradiated SC cells at 48 hrs after their exposure to γ radiation (Figure 6). Results show that
one of the most powerful antioxidant mechanisms related to the NRF2 pathway, which was
clearly activated at 24 hrs (Figures 6 and 7), became silent at 48 hrs post-irradiation (Figure 6).
Moreover, a small down-regulation of a particular NRF2 target was evidenced at all investigated
doses, namely of the SRXN1 gene (FR=-1.47±0.01). SRXN1 encodes sulfireduxin-1, an enzyme
with oxidoreductase and sulfiredoxin activity which confers resistance to oxidative stress by
reducing cysteine-sulfinic acid formed under exposure to oxidants in particular peroxiredoxins
(PRDX 1-4) (Chang et al., 2004). The obtained gene expression data point out towards an
enhanced oxidative and nitrosative stress, deriving from over-expression of genes encoding
ROS/RNS sources and from normal expression or down-regulation of some important
antioxidant mechanisms in irradiated SC cells at 48 hrs post-exposure to γ radiation. At this time
point, a decrease in the number of metabolically active cells and alteration of the plasma
membrane were also registered in irradiated cultures (Figure 5a and 5b, respectively). It is
possible that up- or down-regulation of the mentioned genes, at the low expression changes that
were detected, does not inflict major cellular damages, but are in fact modulating redox signaling
(Forman et al., 2014).
The most striking molecular signature of oxidative activity registered at 48 hrs after exposure of
SC cells to γ radiation was observed at the higher investigated dose of 5 Gy (Figure 6) when
several genes encoding ROS/RNS sources were shown to be up-regulated (genes encoding ROS
sources: NOX5, DUOX1, DUOX2, NCF1, NCF2, MPO; genes encoding RNS sources: NOS2).
Most of these genes address the production of superoxide by NOX enzymes (NOX5, DUOX1,
DUOX2, NCF1, NCF2). Over-expression of genes encoding for ROS generators was not

21
accompanied by a broad up-regulation of cytoprotective NRF2 targets (Figure 6). Moreover, a
marked down-regulation of some other cytoprotective mechanisms was registered, such as those
mediated by the GPX3 gene which encodes for glutathione peroxidase 3 (Figure 8). Glutathione
peroxidase 3 (GPX3) is a secreted isozyme which catalyzes the reduction of organic
hydroperoxides and hydrogen peroxide by glutathione, and thereby provides extracellular
protection against oxidative damage in the microenvironment (Van Brussel et al., 2012). As
such, it appears that SC cells exposed to higher doses of γ radiation were less protected against
the increased radiation-triggered oxidative stress. This accounts for the observed decrease in
MTS reduction and increase in LDH release (Figure 5), indicating a reduced number of
metabolically active cells and alteration of membrane integrity in irradiated cells versus non
irradiated ones.

6.0
5.0
4.0
3.0
Fold regulation (FR)

2.0
1.0
0.0
-1.0 GPX3 NOX5

-2.0
-3.0
-4.0
-5.0
-6.0

Figure 8. Expression of particular redox genes exhibiting significant up- or down-regulation

(-1.5≤ FR >1.5) at all investigated doses (1.25, 2.5 and 5 Gy) in SC monocytes exposed γ rays
delivered at a dose rate of 10 Gy/h. Gene expression was assessed at 48 hrs post-irradiation by
qRT-PCR using a pathway-focused array. Results are expressed as fold regulation (FR) and are
presented as mean ± SD for triplicates.
The expression the BNIP3 and DUSP1 genes which regulate cell proliferation and susceptibility
to apoptosis, as well as autophagy, was not significantly changed at 48 hrs post-exposure of SC
monocytes to γ rays, albeit irradiated cells exhibited decreased MTS reduction (Figure 5a). A
possible explanation might be that cells rely on other regulatory mechanisms than those mediated
by the above mentioned genes. Interestingly, when analyzing the changes in gene expression
during the evolution of non-irradiated cells from 24 hrs to 48 hrs post-exposure to γ radiation
(Table 3) we found another potential reason. In non-irradiated cells, BNIP3 and DUSP1
registered an up-regulation at 48 hrs as compared to 24 hrs, indicating an increased susceptibility
of these cells to apoptosis, as well as a proliferation inhibition probably due to the overgrow of
SC cells in 48 hrs cultures (2.16 x 106 viable cells/mL at 48 hrs versus 0.6 x 106 viable cells/mL

22
at 24 hrs in the same cultures). Moreover, when analyzing the BNIP3 and DUSP1 gene
expression in irradiated cells at 48 hrs post-exposure against the non-irradiated control at 24 hrs
(Figure 10), results showed that gene expression levels were maintained elevated in all irradiated
samples (FR>1.5), irrespective of the causes of this increase (overgrow of cells in non-irradiated
cell cultures or irradiation effects). Similar results were obtained regarding the expression of
various genes encoding ROS sources (DUOX2) and NRF2 targets (HMOX1 and TXNRD1 genes)
which became over-expressed in non-irradiated cells at 48 hrs and masked at this time point a
similar increase potentially triggered by exposure of cells to γ radiation (Table 3). We cannot
rule out that the over-expression of the above mentioned genes might be underlined by distinct
mechanisms in irradiated and non-irradiated SC cells. Altogether these results indicate that there
might be a critical level of gene expression induced by various stressors (in our case cells
overgrow or radiation exposure) that is sensed by cells which further modulate gene expression
accordingly. Of note is the over-expression of the antioxidant GPX3 gene in 48 hrs-cultures of
non-irradiated monocytes and its under-expression in cells exposed to γ radiation at all
investigated doses (Table 3, Figure 9).

Molecular pathway Gene FR Molecular pathway Gene FR

DUSP1 1.45 HMOX1 1.65

Proliferation and cell death
BNIP3 1.98 GCLC 1.73
ALOX12 1.35 GPX2 2.78
Lipid metabolism and
inflammation PTGS2 2.09 GSR 1.46
NRF2 targets
NOX5 -1.20 TXNRD1 1.65
DUOX1 1.28 SEPP1 2.95
DUOX2 1.55 AKR1C2 2.02
ROS sources
NCF1 -1.24 NQO1 -1.42
NCF2 1.15 GPX3 2.48
MPO 1.07 Other antioxidants SOD1 -1.33
RNS sources NOS2* -1.88 SOD2 1.84
Table 3. Gene expression in non-irradiated SC monocytes cultivated for 48 hrs as compared to
gene expression registered in 24 hrs cultures. Gene expression was assessed by qRT-PCR using a
pathway-focused array and results are expressed as fold regulation (FR). Genes with FR values
higher than 1.5 were highlighted in bold.

23
 Gene expression at 48 hrs in irradiated cells
as compared to the 24 hrs CTRL
3.5
CTRL 1.25 Gy 2.5 Gy 5 Gy
3.0
2.5
2.0
1.5
Fold regulation

1.0
0.5
0.0
-0.5 BNIP3 DUSP1 DUOX2 HMOX1 TXNRD1 GPX3
-1.0
-1.5
-2.0
-2.5

Figure 9. Expression of particular redox genes in SC monocytes exposed γ rays (delivered at a

dose rate of 10 Gy/h), analyzed at 48 hrs post-irradiation as compared to non-irradiated cells
(CTRL) at 24 hrs post-exposure. Gene expression was assessed by qRT-PCR using a pathway-
focused array. Results are expressed as fold regulation (FR) of genes at 48 hrs post-exposure
against the expression in non-irradiated cells at 24 hrs.

3.2. The effects of γ radiation delivered at various dose rates

A preliminary study was performed to assess the impact of 1 Gy γ rays, that were delivered at
various dose rates (1.6 Gy/h, 4 Gy/h and 10 Gy/h), on redox genes expression assessed in SC
monocytes at 48 hrs post-irradiation.
For all investigated dose rates, SC monocytes exposed to 1 Gy exhibited decreased MTS
reduction (Figure 10), indicating a decline in the number of metabolically active SC monocytes
in irradiated cell cultures. The most prominent effect was registered at the lowest investigated
dose rate (1.6 Gy/h). Meanwhile, LDH release was not significantly changed in irradiated
samples, pointing out that membrane integrity was not significantly altered by irradiation.

24
 1.6 Gy/h 4 Gy/h 10 Gy/h

Irradiated versusnon-irradiated cells

100

***
x 100

*
**

0
MTS reduction LDH release

Figure 10. MTS reduction and LDH release by SC monocytes exposed to 1 Gy γ radiation
delivered at dose rates of 1.6, 4 and 10 Gy/h. Results were presented as responses of irradiated
versus non-irradiated cells using for calculation the mean of triplicate samples. * p<0.05, **
p<0.01, P<0.0001.
Analysis of the gene expression profile in SC monocytes exposed to γ radiation of 1 Gy that was
delivered at various dose rates (Figure 11) indicated that an enhanced expression of redox genes
in irradiated samples was registered at dose rates of 1.6 Gy/h and 4 Gy/h, but not at the higher
investigated dose rate of 10 Gy/h. The effect on gene expression was most obvious at the lowest
investigated dose rate (1.6 Gy/h) when many genes exhibited expression changes with FR values
higher than 1.5.
The decrease of the number of metabolically active SC cells in irradiated cell cultures at 48 hrs
post-exposure, evidenced by the MTS reduction test (Figure 10), was paralleled by the up-
regulation of the DUSP1 gene (Figure 11). DUSP1 encodes a phosphatase with dual specificity
for tyrosine and threonine, which plays an important role in cellular responses to environmental
stressors, as well as in the negative regulation of cellular proliferation. No correlation could be
established between gene expression and the intensity of the MTS reduction. This is probably
due to the fact that other mechanisms than those mediated by DUSP1 were also acting for
limiting the number of cells in γ-irradiated SC monocytes.
At 48 hrs post-exposure of monocytes to γ radiation several genes that encode ROS/RNS sources
were found up-regulated when γ radiation was delivered at medium and lower dose rates of 1.6
Gy/h and 4 Gy/h, respectively, but not when radiation was delivered at the higher investigated
dose rate of 10 Gy/h (Figure 11). The genes that were found over-expressed in irradiated cells
both at 1.6 Gy/h and 4 Gy/h are presented in Figure 12. An over-expression of genes encoding
for the following superoxide-generating enzymes was registered: NCF1 (FR=2.9±1.1), DUOX1
(FR=1.9±0.2) and NOX5 (FR=2.1±0.7). A potentially increased ability of irradiated SC cells to
produce superoxide was evidenced indirectly by the over-expression of the SOD2 and UCP2
genes which encode protective molecules that are acting to limit excessive levels of superoxide.
Additionally, significant over-expression of the MPO gene was registered (FR=3.7±1.3),
evidencing a complex oxidative pattern of oxidative activity in irradiated cells. MPO encodes
myeloperoxidase which catalyses the generation of the highly toxic hypochlorous acid starting
from hydrogen peroxide. Accordingly, gene expression results indicate that cells exposed to γ
radiation reinforce their ability to generate ROS and to shift the ROS profile from superoxide

25
anion towards more toxic ROS through reactions catalyzed by superoxide dismutases and
myeloperoxidase. Altogether, when radiation was delivered at the dose rates of 1.6 Gy/h and 4
Gy/h, the obtained results are pointing out towards an increased ability of SC cells to generate
ROS at 48 hrs after exposure to γ radiation of 1 Gy,
A potentially increased oxidative activity in irradiated SC cells was also highlighted by the
enhanced molecular signature of the cytoprotective NRF2 transcription factor which is known to
respond to oxidants and electrophiles. A marked expression of several of the NRF2 target genes
(HMOX1, FTH1, NQO1, GSR, SQSTM1, SRXN1 and PRDX1) was found when cells were
irradiated with γ radiation delivered at a lower dose rate of 1.6 Gy/h (Figure 11). The most
striking up-regulation was registered for the HMOX1 gene (FR=7.5) and for the FTH1 gene
(FR=19.2), both genes being involved in iron homeostasis. HMOX-1 encodes heme oxygenase 1,
an inducible isoform, which degrades heme, converts it to biliverdin and triggers the release of
iron and carbon monoxide, hence protecting tissues at various levels against a wide range of
injuries including hypoxic, ischemic and inflammatory disturbances (Lang et al., 2005). FTH1
encodes the ferritin heavy chain 1, a ferroperoxidase which converts ferrous to ferric ion. It is the
major intracellular iron storage protein that regulates and buffers the labile cellular pool of iron,
hence controlling the Haber-Weiss reaction through which the toxic hydroxyl radical is
generated. Moreover, ferritin cooperates with the tumor suppressor p53 to better cope with
oxidative stress (Lee et al., 2009). It is worth mentioning that the observed up-regulation of the
NCF1 gene encoding the p47-phox component of NOX enzymes might have an important role in
NRF2 activation by suppressing NRF2 ubiquitination (Ha Kim et al., 2017), and therefore ROS-
generating enzymes are controlling themselves ROS levels.

26
 Dose rate
Molecular pathway Gene
1.6 Gy/h 4 Gy/h 10 Gy/h
DUSP1
Proliferation/death
BNIP3
Inflammation PTGS2
NCF1
NCF2
ROS sources DUOX1
NOX5*
MPO
RNS sources NOS2*
HMOX1
FTH1
NQO1
GSR
NRF2 targets
SQSTM1
SIRT2
SRXN1
PRDX1
TXN
TXN metbolism
TXNRD2
SOD2
UCP2
Other cytoprotectors
GPX3
HSPA1A
Figure 11. Heatmap chart illustrating the expression level of redox genes in SC monocytes
exposed to γ radiation of 1 Gy, delivered at delivered at various dose rates. Gene expression was
assessed by qRT-PCR at 48 hrs post-irradiation.
Colour code:
FR≥6
4<FR<6
2≤FR≤4
1.5<FR<2
-2<FR<-1.5
-4<FR≤-2
FR≤-4
* genes with very low expression level in non-irradiated SC cells.

27
 9.0

8.0

7.0
Fold regulation (FR)

6.0

5.0

4.0

3.0

2.0

1.0

0.0
DUSP1 NCF1 DUOX1 NOX5 MPO HMOX1

Figure 12. Genes with modified expression in SC cells exposed to γ rays of 1 Gy, delivered at
1.6 Gy/h or 4 Gy/h. Gene expression was assessed by qRT-PCR at 48 hrs post-irradiation.
Results were presented as fold regulation (FR). Only genes which exhibited modified expression
irrespective of the dose rates were shown. Results are presented as mean ± SD for duplicate
samples.

4. DISCUSSION
It is known that γ radiation is rapidly triggering water radiolysis and formation of hydrogen
peroxide, followed by chemical generation of secondary ROS in a time-frame of ∼10−16 s -
10−6 s (Najafi et al., 2014). Nevertheless, the biological consequences of this initial radiation-
triggered oxidative burst may last for days and even months due to the formation of organic
radicals entering in self-sustaining chain reactions, as well as due to a complex array of signaling
events that aim to repair the oxidative damages inflicted by radiation.
The gene expression data obtained by us evidenced that an increased ability of SC monocytes to
develop intracellular oxidative activity is registered relatively late after irradiation, at 24 hrs and
48 hrs post-exposure of SC monocytes to γ rays.
According to our results, the up-regulation of particular genes encoding enzymes that catalyze
the formation of ROS characterizes the response of monocytes exposed to γ rays. Thus, in
various irradiation settings we found over-expression of genes encoding various components of
NADPH-oxidases (NOX) which catalyze the formation of superoxide anion outside of the
oxidative phosphorylation chain in mitochondria (Lambeth and Neish, 2014). Over-expression
was found at the level of the NCF1 and NCF2 genes that encode the cytosolic factors p47-phox
and p67-phox of NOX enzymes. Moreover, an over-expression of the NOX5 gene, that was
lately shown to be constitutively expressed in monocytes (Manea et al., 2015), was also
registered. The newly generated NOX components add to the activity of the already preformed
components which assemble in response to a triggering stimulus to form functional enzymes for
generating superoxide anion (Rada and Leto, 2008). Enhanced production of superoxide anion in
irradiated SC monocytes was also indirectly evidenced in the present study by the over-

28
expression of particular genes that control cellular superoxide levels (SOD2 and UCP2 genes)
and are an indicative of non-physiologically increased levels of superoxide that cells tend to
limit. SOD2 encodes for the mitochondrial superoxide dismutase 2 (manganese-dependent)
which catalyzes the dismutation of superoxide to hydrogen peroxide, while the UCP2 gene
encodes for the mitochondrial uncoupling protein 2 which separates oxidative phosphorylation
from ATP synthesis. In particular irradiation settings, over-expression of NOX genes was
accompanied by the up-regulation of the NOS2 gene encoding for the inducible form of NO
synthase 2 (iNOS) which catalyzes the formation of NO. Superoxide and NO are reacting to
form the toxic peroxynitrate, hence increasing the extent of oxidative damages.
Most interestingly, we also registered the up-regulation of the genes encoding the peculiar NOX
enzymes DUOX1/2 (dual oxidases) that generate hydrogen peroxide. It has been demonstrated
that the levels of DUOX1 and 2 proteins and mRNAs can be induced by NOX-derived
superoxide through redox signaling events, hence explaining their over-expression in the context
of ROS-generating γ rays. A possible mechanism was proposed by Damiano et al. (2012): NOX2
(see above the over-expression of the NCF1 and NCF2 genes) induces the activation of the
mitogen-activated protein kinase ERK1/2 and of the phosphoinositide 3-kinase which activate
NOX enzymes though a feedback loop. The ensuing ROS inhibit tyrosine phosphatases and
maintain ERK1/2 in an active form, ultimately leading to stabilization of specific DUOX1/2
mRNAs and increase of protein levels (Damiano et al., 2012). Additionally, it was demonstrated
that IL-13 in conjunction with the p38 mitogen-activated protein kinase may be also responsible
for persistent DUOX1-induced hydrogen peroxide production following irradiation of thyroid
cells (Ameziane-El-Hassani et al., 2015), indicating that inflammation might be a key regulator
of DUOX enzymes.
The obtained results regarding the over-expression of genes encoding for various components of
NADPH oxidases are in line with other studies showing that NADPH oxidases, including DUOX
enzymes, appear to be critically involved in the persistent oxidative burst triggered by radiation
exposure (Yahyapour et al., 2018).
Besides the up-regulation of genes encoding for NADPH oxidases we have also found the up-
regulation of the MPO gene encoding myeloperoxidase that catalyzes the formation of
hypochlorous acid. Accordingly, results indicate the evolution of ROS from superoxide and
hydrogen peroxide towards more toxic ROS in irradiated monocytes.
The potentially increased oxidative activity in irradiated monocytes, underlined by up-regulation
of genes encoding ROS sources, was indirectly substantiated also by the over-expression of
genes encoding cellular antioxidants, that are controlled at transcriptional level by the
transcription factor NRF2. The activation of NRF2-mediated transcription was most obvious
when SC monocytes were exposed to γ rays of 1 Gy delivered at lower dose rates (1.6 Gy/h).
The HMOX1, NQO1 and SQSTM1 genes, which are the main NRF2 targets, were found
significantly over-expressed (FR>2) in irradiated cells. The involvement of NRF2 in the
response of monocytes to γ irradiation was further confirmed by the moderate over-expression of
several other NRF2 targets such as the GSR, SRXN1 and PRDX1 genes.
Altogether, gene expression changes pointed towards an increased oxidative activity in SC
monocytes exposed to γ rays, which was registered relatively late post-irradiation. The type of
molecular redox events triggered by irradiation in human SC monocytes seems to be dependent
on the dose and dose rate at which γ rays were delivered. Results highlight that major redox

29
responses, most of them cytoprotective, occur at doses around 1 Gy, especially when γ rays are
delivered at lower dose rates. This observation is in line with other studies showing that 1Gy
radioactive exposure seems to be a critical threshold dose that elicits important expression
changes in genes associated with carcinogenesis and metabolic disorders such as FADD,
TNFRSF10B, TNFRSF8, TNFRSF10A, TNFSF10, TNFSF8, CASP1 and CASP4 (Lee et al.,
2014). We emphasize herein that a low redox signature was detected in SC monocytes exposed
to higher doses of γ rays (5 Gy) delivered at the dose rate of 10 Gy/h. Possibly, in this irradiation
setting extensive damages were inflicted to irradiated monocytes by the initial ROS burst
triggered by γ rays, most probably profound DNA lesions, and therefore redox signaling was
shut down. We underline that this study did not aim to perform an in-depth investigation on the
influence of dose and dose rate on the redox signature in monocytes exposed to γ radiation. In
fact, we used various irradiation settings for screening the potential redox changes that may be
elicited in exposed cells.
The increased ability of irradiated SC monocytes to generate ROS, evidenced by gene expression
changes, might contribute to the observed decrease of metabolically active cells in irradiated cell
cultures. Cell death by apoptosis might occur considering the registered up-regulation of the
BNIP3 gene in SC cells exposed to γ rays delivered at 10 Gy/h. BNIP3 is involved in apoptosis
and autophagy, hence contributing to the clearance of damaged cells, especially when important
mitochondrial disturbances are present. As reviewed by Hu et al. (2016) radiation can cause
injury to extra-nuclear targets such as the plasma membrane, mitochondria and endoplasmic
reticulum (ER), hence inducing accumulation of ROS, additional ROS-induced injuries to
mitochondria and ER. This is finally leading to the activation of multiple stress signaling
pathways and of the ER membrane sensors of stress, resulting in apoptosis and/or autophagy (Hu
et al., 2016). Protective autophagy was also evidenced by the registered up-regulation of the
SQSTM1 gene encoding for the ubiquitin-binding protein p62 which is a classical receptor of
autophagy, involved in many signal transduction pathways, including the KEAP1–NRF2
pathway (Liu et al., 2016). The observed over-expression of the DUSP1 gene might be a redox-
regulated mechanism through which γ irradiated cells tend to protect themselves against the
deleterious action of γ irradiation, as evidenced by the decrease of the number of metabolically
active cells in irradiated cultures. DUSP1 encodes for the redox-sensitive dual-specificity
phosphatase MKP-1 which attenuates the activities of p38 and JNK mitogen-activated protein
kinases to induce cell cycle arrest. Particularly in monocytes/macrophages, MKP-1 decreases
priming, migration and recruitment of monocytes (Kim et al., 2012), and regulates the
production of both pro- and anti-inflammatory cytokines (Chi et al., 2006). The redox-sensitive
DUSP1 gene is probably complementing the action of other genes involved in the cell cycle
arrest (Budworth et al., 2012) occurring in cells exposed to ionizing radiation (Fournier and
Taucher-Scholz, 2004).
Phenotypic and functional changes triggered by irradiation, and their connection to the
observed gene expression changes have to be clarified in future studies.

5. CONCLUSION
The expression profile of 84 redox genes points towards an increased oxidative activity in human
SC monocytes exposed to γ rays (1-5 Gy, 1.6-10 Gy/h), which is persisting through endogenous
mechanisms after the initial water radiolysis and ROS burst triggered by γ rays. Increased

30
expression of redox genes was registered in various irradiation settings, addressing ROS/RNS
sources, especially genes involved in superoxide generation, ROS metabolism, ROS targets and
endogenous antioxidants. Irradiated cells develop redox-dependent mechanisms to face cell
death and to arrest the cell cycle for increasing their ability to repair radiation-induced damages.
At lower dose rates, the increased signature of the cytoprotective transcription factor NRF2 was
apparently insufficient to avoid the decrease of the number of metabolically active cells exposed
to γ rays. Moreover, only a low transcriptional activity of NRF2 was registered at higher doses of
γ radiation, at which probably profound oxidative damages occurred. Therefore, pharmacologic
activation of the NRF2 pathway seems to be a promising therapeutic strategy aimed at
counteracting the deleterious effect of γ rays on normal monocytes.

Acknowledgements:
Funding: This work was supported by the Romanian Ministry of Research and Innovations
through the grants 13-ELI/2016 and PCCDI-35/2018.

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HIGHLIGHTS
• 84 redox genes were investigated in human monocytes exposed in vitro to γ radiation;
• Important changes in the redox genes network were registered at 24-48 hrs post-
irradiation;
• Irradiated cells exhibit an enhanced potential to generate superoxide anion;
• NRF2 appears as a promising therapeutic target to protect irradiated normal monocytes.
 THE EXPRESSION PROFILE OF REDOX GENES
IN HUMAN MONOCYTES EXPOSED IN VITRO TO γ RADIATION

Gina Manda1*, Cristian Postolache2, Ionela Victoria Neagoe1, Andreea Csolti1, Elena
Milanesi1, Maria Dobre1

AUTHOR CONTRIBUTION STATEMENT

Gina Manda: conceptualization, formal analysis; Writing - original draft

Cristian Postolache: Investigation – irradiation of cellular samples, dosimetry; Writing –

experimental irradiation settings, dose distribution

Ionela Victoria Neagoe: Investigation – cell culture and cellular systems, processing of
biological samples for various tests; Editing – references

Andreea Csolti: Investigation – viability tests; Editing – tables and figures

Elena Milanesi: Investigation – formal analysis - processing of gene expression data

Maria Dobre: Investigation – assessment of gene expression patterns.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: